Sickle cell anemia results from a defect in the genetic coding of the hemoglobin molecule. The abnormal hemoglobin S molecule in the red blood cells of sickle cell anemia patients is relatively insoluble and prone to deoxygenation-induced polymerization. When the sickle hemoglobin releases its oxygen at peripheral tissue sites, it undergoes aggregation which distorts the red cell's normal biconcave disc appearance and the characteristic spiculated, "holly leaf" or sickle shape is formed. This aberrant cell distortion accounts for the clinical problems in this disease, namely hemolytic anemia and vascular stasis, occlusion and thrombosis.
The viscosity of the red blood cell interior is largely a function of hemoglobin concentration. When cell water content falls, the deformability of the red blood cell decreases. This compromises the ability of the red blood cell to maneuver through the microvasculature of the body. This effect is magnified in the deoxygenated sickle red blood cell because the rate at which the hemoglobin S polymerizes is greatly accelerated by cellular dehydration, because aggregation is extremely dependent upon the deoxy-hemoglobin S concentration. There is also evidence that the sickle cell becomes dehydrated during deoxygenation and this initiates the cell sickling.
Additional research has indicated that a dysfunctional membrane of the sickle red blood cell plays a fundamental roll in disease pathophysiology. Studies indicate that the deformation or sickling process resulting from deoxygenation requires about 10-15 seconds to complete. Thus, it may be necessary for the blood in the microvasculature to slow abnormally before sickling of most cells occurs. This slowing could be induced because of abnormal interactions between the sickle cell and the micro-blood vessel wall due to an altered red blood cell membrane construction. There may also exist subpopulations of abnormally dense and inflexible red blood cells reflecting cellular dehydration and increased internal viscosity.
These features are presumed to be due to altered ion flux and homeostasis controlled by the cell membrane. Furthermore, when a cell sickles and unsickles repeatedly, the membrane is affected and the cell becomes irreversibly sickled, remaining deformed even when the hemoglobin S is reoxygenated in the lungs. These irreversibly sickled cells (hereinafter ISC) have a drastically reduced water content and, thus, high hemoglobin concentrations, as well as high calcium and low potassium content. They may also be ATP-depleted. ISC's have short intravascular life-spans and the severity of the hemolytic process is directly related to the number of these cells in a patient's circulation. Presumably, the permanently deformed cytoskeletons of the ISC's make them unable to negotiate through capillaries.
Based on the above evidence, researchers have sought a drug that would interact with the red blood cell membrane and increase sodium permeability and/or inhibit calcium uptake and potassium chloride plus water loss. Further, by preventing cellular dehydration, the drug would serve to preserve the deformability of the sickle red blood cell, prevent sickling, and be an extremely valuable therapeutic approach to the control of sickle cell anemia crisis.
As a result, a large variety of structurally and mechanistically dissimilar antisickling agents have been proposed for the treatment of sickle cell anemia. So far, however, none of these have been found to be clinically useful. Most inhibit red blood cell sickling either by chemically modifying the hemoglobin S molecule to prevent its aggregation or by increasing red blood cell affinity for oxygen. Approaches include the use of acetylators, such as aspirin; cross-linking agents, such as dimethyladipimidate; inhibitors of intra-molecular hydrophobic bonds, such as urea; and chemical group reactants, such as cyanate (as in U.S. Pat. No. 3,833,724 to Cerami et al).
Because of the critical role of hemoglobin S concentration in the sickling process, it has been proposed that even a modest reduction of mean cell hemoglobin concentration in the sickle cell will greatly decrease any propensity to sickle. Clinical trials in which sickle patients on a low sodium diet were given anti-diuretic hormone to reduce plasma Na.sup.+ -osmolality in hopes of producing a secondary reduction in cell hemoglobin S concentration had very limited success. Low plasma Na.sup.+ levels simply could not be maintained. Further, this is an ineffective means of increasing red blood cell water content. Other investigators have used the antibiotic Monensin to induce a Na.sup.+ -selective leak in the red blood cell membrane. It was found that this Na.sup.+ -ionophore produced an influx of sodium salt and water into the sickle cell. This increased hydration produced some improvemement in the deformability of the red blood cell. Disadvantageously, however, Monensin is highly toxic in experimental animals at micromolar levels.
From the above, it is clear that a need exists for a safe, effective and clinically useful compound for the treatment of sickle cell anemia. During the course of experiments designed to photoaffinity label and identify the glucose transport carrier in normal human red blood cells, we found that phlorizin benzylazide caused hemolysis of the red blood cells under certain conditions. The phlorizin derivative has a high, but reversible, affinity for the sugar transporter in subdued light and is a potent inhibitor of the transfer mechanism. After allowing the compound to associate with its specific membrane receptor, activation with light was expected to result in relatively specific, covalent, irreversible labeling of the transporter. However, in our initial experiments, even before illumination, the red blood cells were found to swell and, in a time and drug dose-dependent manner, burst even in an isotonic buffer. These results suggested to us that this compound has potential usefulness as a membrane perturbant and anti-sickling agent. Further experiments were then conducted to demonstrate the safety and effectiveness of phlorizin benzylazide for use in a method of treating sickle cell anemia.